The goal of this proposal is to bypass the cooperativity of protein folding and stability by dissecting a protein's structure and folding process into smaller pieces. This will be done by isolating and characterizing fragments of the protein that fold in isolation,a nd by identifying and characterizing partially-folded states of the protein. These subdomains and equilibrium intermediates will allow an evaluation of the same polypeptide chain in different contexts. The model system used in the proposed experiments is the ribonuclease H (RNase H) family of enzymes. The recent crystal structures for both E. coli RNase H and the RNase H domain from HIV reverse transcriptase reveal natural examples of subdomains. In addition, preliminary studies have revealed two more subdomains. one 15 residue long helical subdomain and another isolated by limited proteolysis containing approximately half the protein. An equilibrium intermediate state of the protein similar to the "molten globule" has also been uncovered. The proposed experiments describe a thermodynamic and structural analysis of these alternative forms of the polypeptide chain and their relationship to each other. Specifically, the aims of this proposal are: 1. To determine the structure, stability and folding of the peptide fragments, or subdomain(s), obtained by limited proteolysis of RNase H with pronase. 2. To characterize the structure and stability of the helix-forming peptide derived from helix E of the protein. The effect of mutations in this helix on the isolated peptide and the intact protein will be evaluated. 3. To characterize the partially-folded acid state of Ribonuclease H, and to test if a state similar to the partially-folded acid state exists as a transiently populated intermediated in the folding pathway of RNase H.